Environmental Microplastic Exposure Changes Gut Microbiota in Chickens

Simple Summary The harmful effects of microplastic (MP) exposure on aquatic animals have been extensively studied; however, there is a lack of research on its impact on poultry. To address this gap, the present study aimed to evaluate the effects of MP exposure on the growth performance and gut microbiota of chickens. The findings of the study revealed that MPs had a significant negative impact on the growth performance of chickens and can cause an imbalance in gut microbiota. Abstract As novel environmental contaminants, MPs exist widely in the environment and accumulate in organisms, which has become a global ecological problem. MP perturbations of organismal physiology and behavior have been extensively recorded in aquatic animals, but the potential effects of MPs on poultry are not well characterized. Here, we explored the adverse effects of MP exposure on the growth performance and gut microbiota of chickens. Results showed that the growth performance of chickens decreased significantly during MP exposure. Additionally, Firmicutes, Bacteroidota, and Proteobacteria were found to be dominant in the gut microbiota of MP-exposed chickens, regardless of health status. Although the types of dominant bacteria did not change, the abundances of some bacteria and the structure of the gut microbiota changed significantly. Compared with the controls, the alpha diversity of gut microbiota in chickens exposed to MPs showed a significant decrease. The results of comparative analyses of bacteria between groups showed that the levels of 1 phyla (Proteobacteria) and 18 genera dramatically decreased, whereas the levels of 1 phyla (Cyanobacteria) and 12 genera dramatically increased, during MP exposure. In summary, this study provides evidence that exposure to MPs has a significant impact on the growth performance and gut microbial composition and structure of chickens, leading to a gut microbial imbalance. This may raise widespread public concern about the health threat caused by MP contamination, which is relevant to the maintenance of environmental quality and protection of poultry health.


Introduction
The production of plastics has increased faster than any other material over the past few decades, and most plastics are eventually released into the environment [1,2]. Statistically, more than half of the plastics produced globally are used in non-recyclable

Experimental Design
For the animal experiments, a cohort of 60 one-day-old chickens were obtained from a commercial feedlot (Jingzhou, China); these chickens were of similar weight and health status. Standard housing conditions and sufficient diet and water were provided to the chicks to ensure their growth. After three days of acclimatization, the chickens were evenly divided into control (CC) and MP-exposure (MC) groups. The chickens were raised in two cages, with 30 chickens per cage. The control chickens received a normal diet, while the treatment chickens were offered MPs (200 mg/kg) in addition to their normal diet. The MPs provided to chickens were acquired from the Duke Scientific Corporation (product ID CPMS-0.96; Palo Alto, CA, USA); their properties were reported in a previous study [1]. The whole experiment lasted for 28 days, and the dosage of MPs used was based on previous research [33,34]. After the experiment, the chickens were humanely euthanized, and the Animals 2023, 13, 2503 3 of 14 acquired cecal contents were promptly snap-frozen in liquid nitrogen to preserve their integrity for further analysis.

DNA Extraction and Illumine MiSeq Sequencing
The bacterial DNA was extracted from cecal contents of MC and CC groups using a QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) based on the manufacturer's recommendations. Afterward, 0.8% agarose gel electrophoresis and a UV-Vis spectrophotometer (NanoDrop 2000, Waltham, MA, USA) were used to evaluate the integrity and concentration of the extract, respectively. PCR amplification was performed using universal primers (338F: ACTCCTACGGGAGGCAGCA and 806R: GGACTACHVGGGTWTCTAAT) [18,21]. Following the manufacturer's protocol, the purified products were used to construct sequencing libraries using Illumina TruSeq (Illumina, San Diego, CA, USA). The prepared libraries underwent further processing such as purification, quality control, and fluorescence quantification. The libraries that passed the quality inspection and displayed a single peak were considered qualified. Finally, the qualified libraries were diluted, denatured to single-stranded, and then subjected to 2 × 300 bp paired-end sequencing. To acquire the accurate data in subsequent bioinformatics analysis, the original sequences were preprocessed using QIIME software (Qi-ime1.9.1, Flagstaff, AZ, USA). Short sequences (<200 bp), mismatched primers, and chimera were removed. The effective reads were then clustered, and operational taxonomic units (OTUs) were partitioned with a 97% similarity threshold. We generated Venn diagrams to distinguish the number and distribution of OTUs in each group. Prior to performing the bacterial diversity analysis, rank abundance and rarefaction curves were constructed to investigate the sequencing depth. We calculated the microbial diversity of chicken gut microbiota by calculating Chao1, ACE, Shannon, and Simpson indices. To investigate the impact of MP on the gut microbiota of chickens, we generated PCoA plots to assess the gut microbial beta diversity. Differential taxa at different levels related to MP exposure were identified using Metastats analysis and LEfSe. The data are presented as mean ± standard error. Statistical significance was determined as a p value < 0.05.

Growth Performance Analysis
The body weight and average daily weight gain of chickens in the MC group were significantly lower than those in the CC group (Figure 1), whereas there was no significant difference between the MC and CC groups in average daily feed intake. previous research [33,34]. After the experiment, the chickens were humanely euthani and the acquired cecal contents were promptly snap-frozen in liquid nitrogen to prese their integrity for further analysis.

DNA Extraction and Illumine MiSeq Sequencing
The bacterial DNA was extracted from cecal contents of MC and CC groups usin QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) based on the manufacturer's ommendations. Afterward, 0.8% agarose gel electrophoresis and a UV-Vis spectrop tometer (NanoDrop 2000, Waltham, MA, USA) were used to evaluate the integrity concentration of the extract, respectively. PCR amplification was performed using uni sal primers (338F: ACTCCTACGGGAGGCAGCA and 806R: GG TACHVGGGTWTCTAAT) [18,21]. Following the manufacturer's protocol, the puri products were used to construct sequencing libraries using Illumina TruSeq (Illumina, Diego, CA, USA). The prepared libraries underwent further processing such as purifi tion, quality control, and fluorescence quantification. The libraries that passed the qua inspection and displayed a single peak were considered qualified. Finally, the quali libraries were diluted, denatured to single-stranded, and then subjected to 2 × 300 paired-end sequencing. To acquire the accurate data in subsequent bioinformatics an sis, the original sequences were preprocessed using QIIME software (Qiime1.9.1, Flags AZ, USA). Short sequences (<200 bp), mismatched primers, and chimera were remov The effective reads were then clustered, and operational taxonomic units (OTUs) w partitioned with a 97% similarity threshold. We generated Venn diagrams to distingu the number and distribution of OTUs in each group. Prior to performing the bacteria versity analysis, rank abundance and rarefaction curves were constructed to investi the sequencing depth. We calculated the microbial diversity of chicken gut microbiot calculating Chao1, ACE, Shannon, and Simpson indices. To investigate the impact of on the gut microbiota of chickens, we generated PCoA plots to assess the gut micro beta diversity. Differential taxa at different levels related to MP exposure were identi using Metastats analysis and LEfSe. The data are presented as mean ± standard error. tistical significance was determined as a p value < 0.05.

Growth Performance Analysis
The body weight and average daily weight gain of chickens in the MC group w significantly lower than those in the CC group (Figure 1), whereas there was no signifi difference between the MC and CC groups in average daily feed intake.

Data Analysis
In this study, we analyzed 16 cecum samples to compare and investigate changes in the gut microbiota of chickens during MP exposure. We obtained a total of 1,279,763 (CC = 640,238, MC = 639,525) raw sequences, with each sample containing varying raw reads ranging from 79,557 to 80,523 (Table 1)  rarefaction and rank abundance curves demonstrated a saturation trend, suggesting that further increasing the sequencing depth is unnecessary as almost all bacterial species have already been detected (Figure 2A-C). Following taxonomic assignment, these valid sequences were recognized as 627 (CC = 547, MC = 558) OTUs, with the common OTUs in both the CC and MC groups being 100 ( Figure 2D). Furthermore, the numbers of unique OTUs in the CC and MC groups were 69 and 80, respectively. Moreover, the number of OTUs in each sample ranged from 189 to 313 ( Figure 2E). Among the samples, CC1 had the highest quantity of OTUs, while MC8 had the lowest.

Significant Changes in the Gut Microbial Diversity Related to MP Exposure
Good's coverage estimate in each sample was more than 99%, indicating that almost all bacteria could be covered. In addition, the Chao1 (297.06 ± 9.63 versus 255.06 ± 40.38, p = 0.013) and ACE (296.82 ± 9.61 versus 254.89 ± 40.66, p = 0.013) indices were significantly different between the CC and MC groups, while the Simpson (0.98 ± 0.0057 versus 0.97 ± 0.010, p = 0.20) and Shannon (6.86 ± 0.19 versus 6.48 ± 0.50, p = 0.072) indices were not statistically different ( Figure 3A-D). The results of alpha diversity analysis showed that the abundance of gut microbiota in chickens decreased significantly during MP exposure, while the diversity of gut microbiota did not show a significant change. Additionally, the samples from both groups were clearly separated, suggesting significant differences in the major components of the gut microbiota ( Figure 3E,F). These results demonstrate that MP exposure strongly affects the gut microbial alpha and beta diversities in chickens.

Analysis of Gut Microbiota Composition Associated with MP Exposure
To investigate the impact of MP exposure on the gut microbiota, we characterized the compositions and changes of dominant bacterial phyla and genera. Results indicated that a total of 8 phyla and 124 genera were identified, varying from 5 to 8 phyla and from 70 to 99 genera per sample, respectively (Table 2). Specifically, the gut microbiota in CC and MC groups was predominated by Firmicutes (71.74% and 66.89%), Bacteroidota (23.94% and 26.08%), and Proteobacteria (3.17% and 5.90%) in descending order ( Figure 4A). These three dominant phyla accounted for approximately 98% of the total bacterial composition. Other phyla such as Actinobacteriota (0.47% and 0.77%), Desulfobacterota (0.36% and 0.24%), Cyanobacteria (0.19% and 0.06%), unclassified_Bacteria (0.10% and 0.024%), and Patescibacteria (0.0011% and 0.00%) were represented with a lower abundance. Moreover, the dominant genera observed in gut microbiota in the CC group were Bacteroides (23.83%), unclassified_Lachnospiraceae (8.34%), unclassified_Oscillospiraceae (8.03%), and unclassified_Clostridia_UCG_014 (5.43%), whereas Bacteroides (25.46%), unclas-sified_Oscillospiraceae (7.24%), unclassified_Lachnospiraceae (6.97%), and Fournierella (6.80%) were abundantly present in the MC group ( Figure 3B). Additionally, we visualized the clustering heat map to observe the differences in bacterial distribution and variation between the two groups ( Figure 4C). 0.010, p = 0.20) and Shannon (6.86 ± 0.19 versus 6.48 ± 0.50, p = 0.072) indices were not statistically different ( Figure 3A-D). The results of alpha diversity analysis showed that the abundance of gut microbiota in chickens decreased significantly during MP exposure, while the diversity of gut microbiota did not show a significant change. Additionally, the samples from both groups were clearly separated, suggesting significant differences in the major components of the gut microbiota ( Figure 3E,F). These results demonstrate that MP exposure strongly affects the gut microbial alpha and beta diversities in chickens. Beta diversity could be represented by the PCoA scatterplots (E,F). CC: control group. MC: MPexposed groups. All data were represented as mean ± SD. * p < 0.05.

Discussion
The plastic product industry has experienced explosive growth over the past few decades owing to rapid economic development and urban expansion involving many fields of industrial and agricultural production and human life [35]. However, the environmental pollution problems and increased cost of environmental governance caused by the excessive use of plastic products have attracted mounting attention [36,37]. It should

Discussion
The plastic product industry has experienced explosive growth over the past few decades owing to rapid economic development and urban expansion involving many fields of industrial and agricultural production and human life [35]. However, the environmental pollution problems and increased cost of environmental governance caused by the excessive use of plastic products have attracted mounting attention [36,37]. It should be noted that a considerable part of plastic products cannot be recycled but are processed through incineration, deep burial, and discarding which eventually enter the environment and degrade into MPs. The threat of MPs to public health and the health of the animals in husbandry industry has become a prominent issue of concern to many countries and governments. There have been reports on aquatic animals, seabirds, and waterfowl containing MPs, revealing their negative impact on host health [38,39]. The gut microbiota, as the monitor and executor of intestinal function, is inevitably affected by external factors, but information regarding the impacts of MP exposure on gut microbiota in chickens has been scarce. Therefore, we investigated the effects of MP exposure on growth performance and gut microbiota in chickens.
The gut microbiota is naturally stable because of the interaction and plasticity of the microbial community [40]. However, some factors, especially MPs, can disturb the intestinal environment and affect the survival of the microbiota [41]. Under such circumstances, the abundance or type of microorganisms may change to adapt to new intestinal environment, which may lead to the disruption of gut microbial homeostasis. Deng et al. indicated that MP exposure can cause gut microbiota dysbiosis in mice accompanied by metabolic disturbances, increased intestinal permeability, and increased inflammation [42]. Similarly, Sun et al. showed that MP exposure resulted in decreased colonic mucin production, inflammatory responses, and gut microbiota dysbiosis [1]. The indices representing the diversity and abundance include Chao1, ACE, Shannon, and Simpson, which can be used to assess gut microbial homeostasis [43]. Consistent with previous studies, we observed that MP exposure could decrease the Chao1 and ACE indices of gut microbiota in chickens, indicating that MP exposure can decrease gut microbial abundance and induce gut microbial dysbiosis [44]. Maintaining gut microbial homeostasis is crucial for the proper functioning of the intestine, including tasks such as food digestion, nutrient absorption, immune function, and barrier function [45]. However, the perturbation of gut microbial homeostasis may cause various pathological consequences such as intestinal diarrhea, increased intestinal permeability, and metabolic disorders [46,47]. Recent research on gut microbial homeostasis has also revealed its role in the development of diabetes, hypertension, and fatty liver [48]. Therefore, MPs may further cause potential harm to host metabolism, immunity, intestinal function, and health by affecting the homeostasis of gut microbiota. Meanwhile, this may also be one of the reasons for the decreasing growth performance of chickens during exposure to MPs. In addition, we observed significant changes in the major components of the gut microbiota between both the groups. These results demonstrate that gut microbial homeostasis is strongly influenced by MPs.
This study indicated that Firmicutes, Bacteroides, and Proteobacteria were abundant in the gut microbiota of chickens regardless of treatment. These bacteria were demonstrated to be the core components of gut microbiota, which are also abundantly present in ducks, geese, cattle, and pigs [49]. Although the types of dominant phyla did not change, the abundance of some dominant phyla changed dramatically during MP exposure. Proteobacteria, composed of a great deal of Gram-negative bacteria, is the largest phylum in the gut microbiota. Remarkably, some members of Proteobacteria were considered as pathogenic bacteria and opportunistic pathogens, which may seriously threaten host health [50]. In this study, the abundance of Proteobacteria was significantly increased during MP exposure. Thus, MP exposure may result in an increased risk of intestinal disease and other complications in chickens. Previous investigations indicated that environmental MP exposure could significantly change microbial composition and structure [51]. Similarly, the present research also observed significant shifts in gut microbiota of chickens exposed to MPs. Moreover, some significantly changed taxa were regarded as intestinal functional bacteria, which may play crucial roles in intestinal health and homeostasis. Christensenellaceae was considered a potentially beneficial bacterium because of the positive regulation of the hydrolytic enzyme production and intestinal environment [52]. Moreover, Christensenellaceae has been demonstrated to be negatively related to metabolic syndrome, inflammatory bowel disease, and fatty deposits [53]. Notably, some quantitatively decreased bacteria such as Oscillibacter and Blautia were potential producers of short-chain fatty acids (SCFAs). SCFAs have long been deemed as beneficial metabolites due to their vital role in preventing the colonization of pathogens and reducing oxidative stress [54]. Moreover, SCFAs have been shown to possess multiple important biological characteristics such as lowering cholesterol, regulating energy intake, and alleviating inflammation [55][56][57]. Recent investigations on SCFAs also demonstrated their positive impacts in cell proliferation, gut microbial homeostasis, and intestinal barrier function [58][59][60]. Consistent with the current study, MP exposure has also been previously reported to result in a decrease in SCFA-producing bacteria [43]. Importantly, we also found that MP exposure could increase the levels of some pathogenic bacteria, such as Facklamia and Escherichia_Shigella. Facklamia was previously demonstrated to participate in the development of invasive disease such as septicemia and meningitis [61]. Escherichia_Shigella is a potentially pathogenic bacterium associated with increased risk of intestinal infections [62]. Moreover, recently published research about Tyzzerella also indicated that it could drive the development of cardiovascular disease [63]. These bacteria have been demonstrated to play vital role in the balance of gut microbiota. Thus, we speculated that MPs may further affect gut microbial homeostasis by changing these bacteria.
It is well-established that the gut microbiota is a complex micro-ecosystem involving 10 14 micro-organisms, approximately ten times the total quantity of body cells [18]. These microorganisms could interact synergistically or antagonistically to maintain gut microbial homeostasis [64]. Consequently, some changed bacteria may directly or indirectly affect the other bacterial functions, thereby further accelerating gut microbial dysbiosis. In this study, we found significant correlations between some bacteria which may be critical for gut homeostasis. This suggests that MP exposure not only directly affects the microbial composition and structure but also indirectly changes the gut microbiota through the microbial interactions, which may further affect gut microbial homeostasis and amplify the toxic effects of MPs.

Conclusions
In summary, the results of this research support our hypothesis that MP exposure can reduce the growth performance of chickens. Moreover, it also resulted in distinct shifts in gut microbial composition and diversity of chickens. This research is an important exploration of MP exposure on the gut health of farmed animals, suggesting that the imbalance of gut microbiota may be one of the important ways in which MPs lead to ill health.  Institutional Review Board Statement: The animal study was reviewed and approved by the Ethics Committee of the Nanjing Agricultural University (NJAU. No20230413054).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The original sequence data were submitted to the Sequence Read Archive (SRA) (NCBI, USA) with the accession no. PRJNA954763.